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三磷酸腺苷

铁杆木虫 (职业作家)

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应助: 1 (幼儿园)
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性别: GG
专业: 神经系统肿瘤（含特殊感受

[交流] 基于慢病毒的microRNA表达系统-交流转帖

未来一段时间可能想做一个miRNA的稳定转染
所以想找有没有现成的表达载体
自己包装的话挺麻烦的

搜了一下，国外的有Invitrogen、Thermo，国内的有康成（不过似乎也是跟外国公司挂钩的吧）

钱不是问题，要的是效果
不知道有没有人用过，觉得哪个公司好，交流一下～～～

------------------------
8.13
接到版主的奖励通知，呵呵，有点受宠若惊
在此把原来的帖子更名，作为交流专用帖
希望大家能够在这里多多发表，共同交流
比如，什么公司的产品可靠，包装时需要注意什么问题，会不会对哪些细胞产生明显的毒性等等
也不一定专限于miR，其他的基于慢病毒的siRNA表达系统问题也可以在此讨论

我跟Invitrogen要了一些资料，待会我整理完后再跟帖发上来

[ Last edited by 三磷酸腺苷 on 2008-8-13 at 09:28 ]

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1楼 2008-08-10 12:13:33

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三磷酸腺苷

铁杆木虫 (职业作家)

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SBI（System Biosciences）的资料

★ ★ ★ ★ ★
xyklmu(金币+5,VIP+0):thanks

Lenti-miR™ MicroRNA Precursor Clone Collection

http://www.systembio.com/miRNA_Precursor_Collection.htm 原始页面

MicroRNA Precursors expressed from SBI's Lenti-miR™ vectors produce Mature microRNAs

Features and Benefits
Pre-microRNA expression from constitutive CMV promoter
Reliable delivery to dividing or non-dividing cells
Monitor positive expressing cells with copGFP as a reporter under the control of a constitutive human elongation factor 1α (EF1) promoter, which is functional in most cell lines
Convenient sorting of transfected or transduced cells
Pool of prepackaged viral particles containing the entire microRNA Precursor Collection for high-throughput phenotypic screens also available

First microRNA Precursor Collection Available in HIV-based Lentiviral Vectors.
The precursor miRNAs are expressed on average several-fold above endogenous miRNA levels.

Over 400 pre-microRNA constructs in Lenti-miR™ collection
Choose from an extensive collection library or have your pre-microRNA of choice custom built as a service. The collection will be expanded to include all known Human pre-miRNAs.
Download printable list of available pre-microRNA constructs.

Human MicroRNA Precursor ID
Human Precursor Accession #
Catalog #
Price
Control vector
- - -
CD511B-1
$385
let-7a-1
MI0000060
PMIRHlet7a1PA-1
$450
let-7a-2
MI0000061
PMIRHlet7a2PA-1
$450
let-7a-3
MI0000062
PMIRHlet7a3PA-1
$450

……中间省略很多……

miR-99b
MI0000746
PMIRH99bPA-1
$450

Quick Links:
References
Kit Components
Pooled Library
User Manual
Ordering Information
Related Products

Kit Components
Each construct is provided as 1 bacterial stock with the individual construct of choice.
Each kit comes with user manual.

Lenti-miR Virus Library
For added convenience, a pool of prepackaged viral particles containing the entire microRNA Precursor Clone Collection is available.  The pooled library can be used for high-throughput phenotypic screens of microRNA function.

User Manual
User Manual

Ordering Information

Product Description
Catalog#
Size
Price
To Order
Human pre-MicroRNA Expression Construct
PMIRHxxPA-1
Bacterial stock
$450
[url=mailtorders@systembio.com?subject=miRNA Precursor Construct Order]To Order[/url]
Control Vector Plasmid
CD511B-1
10 mg
(plasmid DNA)
$385
[url=mailtorders@systembio.com?subject=miRNA Precursor Control Vector Order]To Order[/url]
Control Vector Prepackaged Virus
CD511VB-1
>107 IFUs
$495
[url=mailtorders@systembio.com?subject=miRNA Precursor Control Vector Order]To Order[/url]
Pooled Library of all pre-MicroRNA Constructs
-
-
-
See Product Webpage

Additional Custom Services (Plasmids & Packaging)

Service Description
Catalog#
Amount
Price (Does Not Include Construct)
To Order
Plasmid DNA Preparation
CS950MR-1
10 mg
$200
[url=mailtorders@systembio.com?subject=miRNA Precursor Construct Plasmid DNA Order]To Order[/url]
Regular Titer Virus of Individual Construct
CS960A-1
>107 IFUs/ml,
>150 ml
$1,295
[url=mailtorders@systembio.com?subject=miRNA Precursor Construct Prepackaged Virus Order]To Order[/url]
High Titer Virus of Individual Construct
CS965A-1
>108 IFUs/ml,
>150 ml
$1,950
[url=mailtorders@systembio.com?subject=miRNA Precursor Construct High Titer Virus Order]To Order[/url]
Ultra-High Titer Virus of Individual Construct for In Vivo Infections
CS970A-1
>109 IFUs/ml,
>150 ml
$2,450
[url=mailtorders@systembio.com?subject=miRNA Precursor Construct Ultra-High Titer Virus Order]To Order[/url]

All microRNA precursors are designed based on miRBase entries.  If your miRNA of interest is not listed above, custom service for precursor expression constructs is available for any miRNA.  [url=mailto

rders@systembio.com?subject=Custom%20miRNA%20Precursor%20Construct%20Inquiry]Inquire about custom precursors.[/url]
[/url] Sanger microRNA database

To obtain more information contact SBI at 888-266-5066 (toll-free in North America) or 650-968-2200, or e-mail to [url=mailto

rders@systembio.com?subject=miRNA%20Library]orders@systembio.com

SBI's first generation FIV-based MiR Precursor Collection

What is unique about SBI’s new MicroRNA Precursor Clone Collection?

First microRNA Precursor Collection Available in HIV-based Lentiviral Vectors
There are expected to be about 1,000 MicroRNAs encoded in the human genome and they function by either blocking translation of, or degrading, mRNA species corresponding to specific genes. While the number of verified human miRNAs is expanding, there is an increasing need for effective functional testing. System Biosciences (SBI) is offering an extensive collection of MicroRNA precursors in lentiviral vectors that can be used to modulate the expression of the cognate MicroRNAs in vivo, and thereby study MicroRNA function. The constructs are built in a lentiviral vector backbone, so they can be packaged into lentiviral particles and delivered to primary cells, stem cells, or other hard-to-transfect cell lines.
System Biosciences (SBI) is the only commercially-available collection of MicroRNAs that are cloned in Lentiviral vectors capable of being propagated. The advantage of SBI’s constructs is that they can be stably expressed in a wide variety of cell types, as opposed to synthetic MicroRNAs that can only be transiently expressed in cells. Moreover, the inserts in the SBI’s microRNA Precursor Collection represent more than just the mature microRNA sequences listed in Sanger’s miRBase (http://microrna.sanger.ac.uk/sequences/). Each construct in SBI’s collection consists of the stem loop structure and 200-400 base pairs of upstream and downstream flanking genomic sequence. This unique feature ensures that the microRNAs expressed from SBI’s constructs would be correctly processed in the cell into mature microRNA.

Details of the Lentiviral Delivery System:
·       Express native microRNAs in target cells through transfection (plasmid form) or transduction (packaged
   constructs).
·       Simple packaging into pseudoviral particles with SBI’s pPACK Lentivector Packaging System
·       Deliver microRNA expression constructs into wide range of cell lines including non-dividing or difficult-
   to-transfect cell lines with biologically-safe and high-titer enabled HIV-based lentivector.
·       RNA polymerase II driven primary microRNA expression offers high level of expression from single copy of
   insert.
Related Products

QuantiMir™ miRNA Profiling Kit
Global MicroRNA Amplification Kit™
MicroRNA Discovery™ Kit
Cancer MicroRNA Profiling Panel
Stem Cell MicroRNA Profiling Panel
HT 384 MicroRNA Profiling System
HT 419 MicroRNA Profiling System

References
miRNA discovery

Lee RC, Feinbaum RL, Ambros V. “The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14”. Cell, 75:843-854, 1993.
Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvits HR, Ruvkun G. “The 21-nucleotides let-7 RNA regulates developmental timing in Caenorhabditis elegans”. Nature, 403:901-906, 2000.
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. “Identification of novel genes coding for small expressed RNAs”. Science, 294:853-858, 2001.
microRNA targets prediction
Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP. “Prediction of plant microRNA targets”. Cell, 110:513-520, 2002.
Lewis BP, Burge CB, Bartel DP. “Conserved seed pairing, often flanking by adenosines, indicates that thousands of human genes are microRNA targets”. Cell, 120:15-20, 2005.
John, B., C. Sander and D. S. Marks (2006). "Prediction of human microRNA targets." Methods Mol Biol, 342: 101-13.
Vector-based miRNA expression
Mathijs Voorhoeve, P., C.L. Sage, M. Schrier, A.J.M.Gillis, H. Stoop, R.Nagel, Y. Liu, J.V.Duijse, J. Drost, A. Griekspoor, E. Zlotorynski, N. Yabuta, G. D.Vita, H. Nojima, L.H.J.Looijenga, and R. Agami (2006). “A Genetic Screen Implicates miRNA-372 and miRNA-373 As Oncogenes in Testicular Germ Cell Tumors.” Cell, 124, 1169-1181
Shin, K. J., E. A. Wall, J. R. Zavzavadjian, L. A. Santat, J. Liu, J. I. Hwang, R. Rebres, T. Roach, W. Seaman, M. I. Simon and I. D. Fraser (2006). "A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression." PNAS U S A, 103(37): 13759-64.
Stegmeier, F., G. Hu, R. J. Rickles, G. J. Hannon and S. J. Elledge (2005). "A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells." PNAS U S A, 102(37): 13212-7.
Primary miRNA processing

Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN. “The Drosha-DGCR8 complex in primary micorRNA processing”. Gene Develop, 18: 3016-3027, 2004.
Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, Sohn SY, Cho Y, Zhang BT, Kim VN. “Molecular basis for the recognision of primary microRNAs by the Drosha-DGCR8 complex”. Cell, 125: 887-901.2006.
Zeng Y, Yi R, Cullen BR. “Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha”. EMBO J, 24:138-148, 2005.
Zeng Y, Cullen BR. “Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences”. J Bio Chem, 280:27595-27603, 2005.
miRNA functional study

Bennasser, Y., S. Y. Le, M. L. Yeung and K. T. Jeang (2004). "HIV-1 encoded candidate micro-RNAs and their cellular targets." Retrovirology, 1(1): 43.
Esau C, Kang X, Peralta E, Hanson E, Marcusson EG, Ravichandran LV, Sun Y, Koo S, Perera RJ, Jain R, Dean NM, Freier SM, Bennett CF, Lollo B, Griffey R. MicroRNA-143 regulates adipocyte differentiation.
J Biol Chem. 2004 Dec 10;279(50):52361-5.
Hariharan, M., V. Scaria, B. Pillai and S. K. Brahmachari (2005). "Targets for human encoded microRNAs in HIV genes." Biochem Biophys Res Commun, 337(4): 1214-8.
Johnson, S. M., H. Grosshans, J. Shingara, M. Byrom, R. Jarvis, A. Cheng, E. Labourier, K. L. Reinert, D. Brown and F. J. Slack (2005). "RAS is regulated by the let-7 microRNA family." Cell, 120(5): 635-47.
Kim, V. N. (2005). "Small RNAs: classification, biogenesis, and function." Mol Cells, 19(1): 1-15.
Lee, R. C., R. L. Feinbaum and V. Ambros (1993). "The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14." Cell, 75(5): 843-54.
Lee, Y., K. Jeon, J. T. Lee, S. Kim and V. N. Kim (2002). "MicroRNA maturation: stepwise processing and subcellular localization." Embo J, 21(17): 4663-70.
Olsen, P. H. and V. Ambros (1999). "The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation." Dev Biol, 216(2): 671-80.
Pfeffer, S., M. Zavolan, F. A. Grasser, M. Chien, J. J. Russo, J. Ju, B. John, A. J. Enright, D. Marks, C. Sander and T. Tuschl (2004). "Identification of virus-encoded microRNAs." Science, 304(5671): 734-6.
Yi, R., Y. Qin, I. G. Macara and B. R. Cullen (2003). "Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs." Genes Dev, 17(24): 3011-6.